The molecular basis of extracellular matrix deposition has been a subject of great interest in recent years. As the molecular components have become known, attention has shifted toward developing pharmaceutical and therapeutic interventions for controlling particular steps in the relevant deposition processes. One critical step is the production of mature triple helical fibrillar collagen, Types I-III. These collagen molecules are synthesized as procollagen precursors that contain amino-terminal (N-) and carboxyl-terminal (C-) propeptides that are cleaved extracellularly to yield the mature triple helical collagen monomers that can associate into fibrils that can themselves be deposited in a matrix (for a review, see Ref. 5). The N- and C-terminal cleavages are accomplished by distinct enzymatic activities, that have been denoted procollagen C-proteinase (PCP) (8-10) and procollagen N-proteinase (PNP) (47).
It would be desirable, for example in the field of wound healing, to modulate the kinetics of collagen deposition. One promising mechanism for doing so is to alter collagen maturation by modulating the activities of PCP and PNP. The efforts of pharmaceutical developers have been hampered by the lack of an effective system for assaying the modulating effects of putative agents that affect PCP and PNP activities. Before therapeutic development can proceed in an in vivo system, an in vitro system is needed that can determine both whether an agent is effective for modulating the maturation of procollagen to collagen and whether the agent is itself toxic to fibrogenic cells.
Recently, the bone morphogenetic protein-1 (BMP-1) was shown to be identical to procollagen C-proteinase (PCP)(6,7), the activity that cleaves the C-propeptides of procollagen types I-III (8-10). The mammalian BMP1 gene that encodes BMP-1 also produces an alternatively spliced mRNA that encodes mammalian tolloid (mTld), a longer protein that has a domain structure identical to that of Drosophila Tld (4). mTld also has PCP activity (7). The necessary action of BMP-1 and mTld in processing of matrix components (6,7,12) and lysyl oxidase (11) suggests that these proteins play key roles in controlling the deposition of matrix in developmental and homeostatic processes. Previously, however, mechanisms for regulating functional expression of these key proteins have not been explored.
BMP-1 copurifies from osteogenic bone extracts with transforming growth factor-.beta. (TGF-.beta.)-like proteins BMP-2 through -7 (1). Thus, it was suggested that BMP-1, by structure an astacin-like protease, may function in morphogenesis by activating TGF-.beta.-like molecules (1). Consistent with this possibility, BMP-1 has a domain structure similar to, but shorter than that of tolloid (Tld), a Drosophila protein that appears to act in patterning of embryos by potentiating the activity of decapentaplegic, a TGF-.beta. family member (2,3).
PCP activity of BMP-1 is stimulated .about.10-fold by the procollagen C-proteinase enhancer (PCPE), a glycoprotein that binds the type I procollagen C-propeptide (10). However, possible involvement of PCPE in other biological activities of BMP-1 and mtld has not been examined.
TGF-.beta.1, prototype of the TGF-.beta. superfamily, induces net increases in deposition of insoluble matrix by cells. This is accomplished by effecting decreased production of proteases that degrade matrix, and increased production of i) inhibitors for such proteases, ii) structural matrix components such as procollagen types I-III (13), and iii) lysyl oxidase (14). Expression of the genes for the three polypeptide chains of laminin 5 is also upregulated by TGF-.beta. in keratinocytes (15).